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New multifunctional coli bacillus nonfusion protein expressing carrier and its ase

A technology of Escherichia coli and protein expression, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as waste of time, heavy workload, success rate, and poor reliability

Inactive Publication Date: 2003-11-26
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

[0004] When screening recombinants of exogenous genes and expression vectors, except for the pETBlue-1 expression vector newly developed by Novagen, which adds blue and white screening methods, the method of extracting plasmids from multiple strains is currently used for identification, which is a waste of time and work. The volume is heavy, and the success rate and reliability are relatively poor

Method used

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  • New multifunctional coli bacillus nonfusion protein expressing carrier and its ase
  • New multifunctional coli bacillus nonfusion protein expressing carrier and its ase
  • New multifunctional coli bacillus nonfusion protein expressing carrier and its ase

Examples

Experimental program
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Embodiment

[0020] Example: 1. In this example, the pGEM7Zf(+) cloning vector is used as the starting plasmid to construct the expression vector A. Materials

[0021] Escherichia coli DH5α, Escherichia coli JM109(DE3), plasmids pGEM7Zf(+) and pGEMEX-2 (see figure 2 ) was purchased from Promega, USA,

[0022] Restriction endonuclease and T4 DNA ligase were purchased from Promega Company in the United States, medium molecular weight protein marker, and DNA column gel recovery kit were purchased from Shanghai Huashun Bioengineering Company. B. Build process

[0023] The construction steps of the expression vector are as follows: Figure 4 shown.

[0024] According to the base sequence of T7 terminator, design a set of primers:

[0025] T7 F: 5'ACAAgCTTCCACCgCTgAgCAATAAC3',

[0026] T7 R: 5′-ACgAgcTCTATAgTTCCTC3′

[0027] Using pGEMEX-2 as a template, the T7 terminator sequence was amplified. After the gel was recovered, double-digested with SacI and HindIII. Simultaneously double-cut...

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Abstract

The present invention is a new kind of prokaryotic organism non-fusion protein expression carrier constructed by means of DNA recombination technology. The present invention features that mainly pGEMcloning carrier is used as initial plasmid, and the SD sequence of T7 phage gene 10 and T7 terminator sequence are added to the two ends of the polycolonal site, and thus there are various elements of expression carrier. ATG newly introduced to NdeI site provides initial codon ATG for expressing required natural protein. The distance between ATG and the 6-12 base in SD conserved sequence is the optimal distance for efficient initial translation. The recombinant may be selected based on blue-white reaction. The cloning, sequencing and expression of foreign gene may be performed in the same carrier.

Description

technical field [0001] The invention relates to an expression vector used in DNA recombination technology in the technical field of genetic engineering, in particular to an E. coli non-fusion protein expression vector and its construction method and application. Background technique [0002] Escherichia coli expression system has the advantages of short growth period, simple operation and high expression level. According to different purposes, the expression vectors can be divided into fusion protein expression vectors and non-fusion protein expression vectors. For proteins used as medicines and for structural and functional studies, in order not to affect their natural properties, their expression products must be completely identical to natural proteins, rather than fusion proteins. Therefore, non-fusion protein expression vectors are of great significance both theoretically and practically. [0003] At present, people generally use the PCR method to obtain exogenous gen...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/70
Inventor 吕安国时成波吴文芳杨立泉
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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